Recombinant proteins can be expressed in different types of host cells including prokaryotic and eukaryotic cells. However, glycoproteins, such as antibodies and Fc fusion proteins, consist of a polypeptide linked to a carbohydrate moiety which influences the safety and efficacy thereof, and thus they are usually expressed in animal cells that are capable of glycosylation during post-translational modification. In the production of recombinant protein drugs, the difference in their sugar chains with those of human (native) glycoproteins is associated with immunogenicity of the protein drugs. Thus, it is important to produce glycoproteins having sugar chains identical or similar to those of human glycoproteins.
Until recently, animal cells such as hybridoma, mouse myeloma, and CHO cells have been commonly used in the expression and production of recombinant protein drugs. The protein expression in these animal cells is appropriate for producing proteins similar to human proteins, but there are disadvantages of a significantly low expression yield and difficulty in scale-up of the production. In particular, therapeutic antibodies need to be produced in kilogram quantities, and thus animal cell culturing is not suitable for a large-scale production of the therapeutic antibodies. Therefore, for a high level expression of a target gene in the host cells, the target gene needs to be integrated into a transcriptionally active region of the genome when the target DNA is randomly introduced to the animal cell. The introduced foreign gene replicates along with the genome of the host cell. However a homologous recombination technique for integration of the gene into transcriptionally active regions is not generalized for common use yet.
Another method for increasing the expression rate of randomly integrated DNA is by amplifying the integrated gene, and this method needs the step of cloning the gene into the vector engineered with gene amplification system. A DHFR system (Takeshi omasa, gene amplification and its application in cell and tissue engineering, J. Bios. and Bioe (2002), Vol. 94, No. 6, 600-605) is a common gene amplification system, and this system increases the protein expression level by co-amplification of a target gene and DHFR using methotrexate (MTX) which is an inhibitor of dihydrofolate reductase (DHFR). DHFR is an enzyme involved in nucleotide biosynthesis, and thus inhibition of DHFR activity can effectively interrupt DNA synthesis which is essential for cell maintenance, thereby leading to cell death. Therefore, only those clones having exogenous DHFR gene inserted in their genome can survive under this condition. Furthermore, when the concentration of MTX being added is increased and a strong promoter is used, DHFR gene can be amplified to hundreds to thousands of copies. That is, the more MTX, a DHFR inhibitor, is added to the cell culture, in order for them to survive they increase the expression of DHFR along with the introduced target gene. Consequently, several copies of DNA will be incorporated, leading to the generation of various molecular variants.
A DHFR system using CHO cell line has been reported and commercialized as an expression system for various protein drugs, verifying its safety and efficacy in use. However, the DHFR system has a disadvantage in that it requires several months to isolate a single cell line that shows the expression level higher than the normal level. In addition, when the cell becomes resistant to MTX, even with an increase in MTX concentration, the target gene cannot be amplified anymore. Furthermore, in the CHO DUKX cells used for DHFR system, revertants may appear easily. As a result, there has been a high demand for the development of a high-level gene expression system for protein production other than the DHFR system.
GS system is a high-level gene expression system that was first developed by Celltech (U.S. Pat. No. 5,122,464), and it overcame the limitations of the DHFR-based gene expression system, that is, low time-efficiency for isolating the single cell line of interest and low productivity of target protein. The GS system utilizes glutamine synthetase (GS) which is an enzyme involved in the sole synthetic pathway for producing glutamine from glutamate and ammonia, based on the fact that animal cells cannot grow properly in the glutamine-deficient condition. The GS system has advantage in that it requires less number of DNA copies per cell compared to the DHFR system and allows for the selection of single cell line having high expression rate at the early stage of screening. Consequently, an increasing number of organizations adopt the GS system as a protein drug expression system. NS0 cell line and CHO cell line are the most commonly used cell lines for GS system. Between two, NS0 cell line which is a mouse myeloma cell line cannot express sufficient amount of GS, and thus in the glutamine-deficient condition, those cells where the target gene is inserted into their genome can be easily selected. Unlike the NS0 cell line, a CHO cell line can express sufficient amount of GS that they can survive even in the glutamine-deficient medium. However, if the CHO cells are treated with a high concentration of GS-specific inhibitor such as methionine sulphoximine (MSX), the cells cannot survive only with the endogenous GS activity, and thus only those cells introduced with the vector comprising the GS gene and the gene for a target protein can survive. Through the above mechanism, the cells inserted with the gene for target protein can be isolated, and the target protein can be produced at high yield. In other words, as more GS-specific inhibitor is added to the cells, in order for them to survive, they will amplify exogenous GS gene as well as the target gene which is introduced together with the GS gene, thereby increasing the amount of target protein in the cell. However, even this GS system has a limitation in production amount when the cells transfected with the vector comprising the genes for target protein and GS are treated with the GS-specific inhibitor for producing the target protein. Also when the cells were cultured for a long time, the production amount of the target protein was reduced. Due to these limitations, there has been a high demand for the development of a modified GS protein that responses more sensitively to the GS inhibitor and thus can amplify the target gene introduced with GS to the greater level.